Abstract:

When a pharmaceutical composition for preventing and/or treating
peripheral neuropathy caused by chromosomal microtubule inhibition, which
comprises erythropoietin as an active ingredient, is administered to
patients receiving a microtubule inhibitor as an anticancer agent, such
treatment alleviates peripheral neuropathy in the patients and enables
increased dosage, prolonged period and increased frequency for
administration of the above anticancer agent, which were never before
achieved. Thus, the pharmaceutical composition not only contributes to
improvement of QOL in the patients, but also enables prolongation of life
in the patients.

Claims:

2. The pharmaceutical composition according to claim 1, wherein the
peripheral neuropathy caused by microtubule inhibition is that caused by
administration of at least one member selected from vinca alkaloids or
taxans.

3. The pharmaceutical composition according to claim 2, wherein a member
of the taxans is paclitaxel or taxotere.

4. Use of an erythropoietin-containing pharmaceutical composition for the
purpose of cancer therapy, wherein the pharmaceutical composition is used
in combination with an anticancer agent having an inhibitory effect on
microtubules.

5. A method for preventing or treating peripheral neuropathy caused by
microtubule inhibition, which comprises administering a pharmaceutical
composition containing a prophylactically or therapeutically effective
amount of erythropoietin to a patient in need of such prevention or
treatment.

6. A method for cancer therapy, which comprises administering to a patient
a pharmaceutical composition containing erythropoietin in an amount
effective for preventing or treating peripheral neuropathy caused by
microtubule inhibition, wherein the pharmaceutical composition is
administered in combination with an anticancer agent having an inhibitory
effect on microtubules.

7. Use of erythropoietin in the manufacture of a pharmaceutical
composition for preventing or treating peripheral neuropathy caused by
microtubule inhibition.

8. A combined formulation, which comprises a pharmaceutical composition
containing erythropoietin in an amount effective for preventing or
treating peripheral neuropathy caused by microtubule inhibition and an
anticancer agent having an inhibitory effect on microtubules.

[0002]Cisplatin and derivatives thereof, as well as taxan-based anticancer
agents have excellent therapeutic effects and are widely used in clinical
practice. However, these agents also cause serious side effects, i.e.,
anemia and peripheral neuropathy. Peripheral neuropathy is a disease with
symptoms of pain, which causes degeneration of nerve axons and/or myelin
disruption. It is well known that peripheral neuropathy is not only
caused by treatment with anticancer agents, but also occurs as a
complication of HIV infection or diabetes.

[0003]On the other hand, erythropoietin (EPO) has been regarded as a
hematopoietic cytokine. Recent reports have indicated that EPO is also
produced in the central nervous system (Masuda et al., 1994), is involved
in the development of the nervous system (Liu et al., 1994; Juul et al.,
1998; 1999; Chin et al., 2000) and shows a wide neuroprotective effect in
diseases such as brain ischemia (Sakanaka et al., 1998; Briness et al.,
2000), spinal cord injury (Gorio et al., 2002), encephalitis (Agnello et
al., 2002), etc.

[0004]Moreover, the presence of EPO and EPO receptors in the peripheral
nervous system has also been reported in rats (Campana & Myers, 2001) and
human tissue (Hassan et al., 2004). This suggests that EPO also plays a
physiological role in the peripheral nervous system.

[0005]As supporting evidence for physiological functions of EPO in the
peripheral nervous system, the group of Campana et al. has reported that
when sciatic nerves of rats are injured or crushed by chronic
compression, EPO expression is increased in glia cells (Schwann cells)
near the injury site (Li et al., 2005). When injured, the sciatic nerves
undergo Wallerian degeneration (axonal degeneration) on the distal side,
while the nerve segment proximal to the injury site serves as a locus for
neurite sprouting (Ide, 1996; Cheng & Zochodne, 2002). As a role of
Schwann cells in peripheral neuropathy, it is known that Schwann cells
are redifferentiated and/or proliferated, and further migrate in the
injury site and its distal side, thereby playing an essential role as a
scaffold for sprouting of new neurites or regenerated axons (Hall &
Gregson, 1974; Ide, 1996; Li et al., 2005). Schwann cells are glia cells
responsible for myelin formation in peripheral nerves and hence
correspond to oligodendrocytes in the central nervous system. The
inventors of the present invention have reported that EPO is a molecule
involved in oligodendrocyte maturation and myelination, and may stimulate
remyelination in demyelinating disease of the central nervous system
(Sugawa et al., 2002; International Patent Publication No. WO02/02135
A1). These findings suggest that EPO acts not only on nerve cells in the
central nervous system, but also on Schwann cells, and may be involved in
nerve protection and/or nerve regeneration and repair during peripheral
neuropathy.

[0007]Moreover, it has been clinically reported that the neurological
symptom scores of cisplatin-treated cancer patients were significantly
improved in the EPO-treated group (Mangiameli et al., 2002).

[0008]However, cisplatin-induced peripheral neuropathy is due to radical
generation, and it is completely unknown whether EPO has a prophylactic
or therapeutic effect on other types of peripheral neuropathy with
different onset mechanisms. In particular, there is no report about the
effects of EPO on peripheral neuropathy induced by a microtubule
inhibitor, when administered as an anticancer agent, which kills cancer
cells by stopping the action of chromosomal microtubules having an
important contribution to cell division.

[0029]The object of the present invention is to provide a pharmaceutical
composition for preventing and/or treating peripheral neuropathy, more
particularly a pharmaceutical composition for preventing and/or treating
peripheral neuropathy caused by microtubule inhibition.

Means for Solving the Problems

[0030]The inventors of the present invention have found that
erythropoietin has a significant therapeutic effect on peripheral
neuropathy caused by a microtubule inhibitor (e.g., taxotere) when
administered as an anticancer agent. This finding led to the completion
of the present invention.

[0031]Namely, the present invention provides the following.

(1) A pharmaceutical composition for preventing and/or treating peripheral
neuropathy caused by microtubule inhibition, which comprises
erythropoietin as an active ingredient.(2) The pharmaceutical composition
according to (1) above, wherein the peripheral neuropathy caused by
microtubule inhibition is that caused by administration of at least one
member selected from vinca alkaloids or taxans.(3) The pharmaceutical
composition according to (2) above, wherein a member of the taxans is
paclitaxel or taxotere.(4) Use of an erythropoietin-containing
pharmaceutical composition for the purpose of cancer therapy, wherein the
pharmaceutical composition is used in combination with an anticancer
agent having an inhibitory effect on microtubules.(5) A method for
preventing or treating peripheral neuropathy caused by microtubule
inhibition, which comprises administering a pharmaceutical composition
containing a prophylactically or therapeutically effective amount of
erythropoietin to a patient in need of such prevention or treatment.(6) A
method for cancer therapy, which comprises administering to a patient a
pharmaceutical composition containing erythropoietin in an amount
effective for preventing or treating peripheral neuropathy caused by
microtubule inhibition, wherein the pharmaceutical composition is
administered in combination with an anticancer agent having an inhibitory
effect on microtubules.(7) Use of erythropoietin in the manufacture of a
pharmaceutical composition for preventing or treating peripheral
neuropathy caused by microtubule inhibition.(8) A combined formulation,
which comprises a pharmaceutical composition containing erythropoietin in
an amount effective for preventing or treating peripheral neuropathy
caused by microtubule inhibition and an anticancer agent having an
inhibitory effect on microtubules.

ADVANTAGES OF THE INVENTION

[0032]As shown in the Example section described later, in taxotere-induced
peripheral neuropathy model mice, EPO was observed to alleviate
peripheral neuropathy both in evaluation of neurological symptoms and in
histopathological evaluation of sciatic nerves.

[0033]Thus, the prophylactic and/or therapeutic agents of the present
invention are useful for peripheral neuropathy and particularly useful
for reducing side effects of peripheral neuropathy in patients receiving
a microtubule inhibitor as an anticancer agent. As a result, the agents
of the present invention not only alleviate pain in the patients
(improves their QOL), but also enable increased dosage, prolonged period
and increased frequency for administration of the anticancer agent, which
were never before achieved. The agents also enable prolongation of life.

[0036]FIG. 3-1 presents photographs showing histopathological changes in
degeneration of sciatic nerve axons and the effect of EPO administration
thereon in the group not receiving taxotere (0 iv), as analyzed by
toluidine blue staining.

[0038]The erythropoietin used in the present invention can be any EPO, but
preferably highly purified EPO, more specifically mammalian EPO,
especially having biological activity substantially identical to that of
human EPO.

[0041]For the purpose of the present invention, fusion proteins of EPO and
another protein can also be used. Fusion proteins can be prepared by,
e.g., ligating the DNA encoding EPO in-frame with the DNA encoding
another protein, inserting the ligation product into an expression vector
and expressing it in a host. The second protein to be fused to EPO in the
present invention is not specifically limited.

[0042]For the purpose of the present invention, chemically modified EPO
can also be used. Examples of chemically modified EPO include, for
example, EPO chemically modified with polyethylene glycol or the like
(e.g., WO90/12874), carbohydrate-free EPO chemically modified with
polyethylene glycol or the like, as well as EPO conjugated to a compound
such as an inorganic or organic compound, e.g., vitamin B12, etc.

[0043]Moreover, for the purpose of the present invention, EPO derivatives
can also be used. As used herein, EPO derivative refers to EPO containing
a modified amino acid in the EPO molecule or to EPO containing a modified
carbohydrate chain in the EPO molecule.

[0044]Modifications of carbohydrate chains in EPO molecules include
addition, substitution, deletion and the like of carbohydrate chains.
Preferred carbohydrate modifications in the present invention include
deletion of sialic acids in EPO molecules.

[0045]Normally, both of EPO produced by recombinant animal cells and EPO
derived from urine are obtained as EPO compositions containing various
EPO molecules having different carbohydrate structures. The number of
sialic acids attached to the EPO molecules in the EPO compositions
depends on the specific EPO molecules, but normally 11 to 15 sialic acids
are attached to one EPO molecule. Desialylated EPO (asialoEPO) can be
prepared by removing these sialic acids. The number of sialic acids
removed by desialylation is not specifically limited, and all sialic
acids may be removed, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14
sialic acids may be removed. Preferred asialoEPO in the present invention
has 10 or less, more preferably 5 or less, most preferably 2 or less
sialic acids attached to the EPO molecule. It should be noted that the
number of sialic acids used in the present invention is the average
number in EPO molecules contained in EPO compositions. The average number
of sialic acids per molecule can be determined by methods known to those
skilled in the art (EPO428267, etc.).

[0046]Desialylated EPO (asialoEPO) can be prepared by methods known to
those skilled in the art, e.g., by treating EPO with an enzyme such as
sialidase. Sialidases are commercially available (JPA 2005-507426, Nobuo
Imai et al., Eur. J. Biochem, 194, 457-462 (1990), etc.).

[0047]Moreover, the erythropoietin of the present invention may be EPO
with a modified carbohydrate chain, including EPO analogs whose
carbohydrate chain is modified, as exemplified by NESP (Novel
Erythropoietin Stimulating Protein; disclosed in WO85/02610, WO91/05867,
WO95/05465, etc.) which has sialic acid attached at the N-terminal end of
EPO.

[0048]Modifications of amino acids in EPO molecules include carbamylation,
biotinylation, amidination, acetylation, guanidinylation, etc., but a
preferred amino acid modification in the present invention is
carbamylation.

[0055]Certain amino acids can also be included as stabilizers (e.g., JPA
HEI 10-182481, etc.). Amino acids added as stabilizers include free amino
acids and salts thereof such as sodium salts, potassium salts,
hydrochlorides, etc. Amino acids can be added alone or as a combination
of two or more. Amino acids added as stabilizers are not specifically
limited, but preferred amino acids include leucine, tryptophan, serine,
glutamic acid, arginine, histidine and lysine.

[0061]anionic surfactants, e.g., alkyl sulfates having a C10-18 alkyl
group such as sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl
sulfate; polyoxyethylene alkyl ether sulfates having an average EO mole
number of 2-4 and a C10-18 alkyl group such as sodium polyoxyethylene
lauryl sulfate; alkyl sulfosuccinic acid ester salts having a C8-18 alkyl
group such as sodium laurylsulfosuccinate; and

[0062]natural surfactants, e.g., lecithin; glycerophospholipids;
sphingophospholipids such as sphingomyelin; sucrose fatty acid esters of
C12-18 fatty acids. Formulations of the present invention can contain one
or more of these surfactants in combination. Preferred surfactants are
polyoxyethylene sorbitan fatty acid esters such as Polysorbate 20, 40, 60
or 80, especially Polysorbates 20 and 80. Polyoxyethylene
polyoxypropylene glycols such as poloxamers (e.g., Pluronic F-68®)
are also preferred.

[0066]In the prophylactic and/or therapeutic agents of the present
invention, the effective dosage of erythropoietin is determined by
physicians in consideration of the disease to be treated and its
condition, the age and body weight of a patient, the route of
administration, etc. In general, EPO is administered at a dose of 0.1 to
5000 μg, preferably 5 to 1000 μg per adult.

[0067]Although the prophylactic and/or therapeutic agents of the present
invention are generally administered by the parenteral route, for
example, in the form of injections (e.g., subcutaneous, intravenous or
intramuscular injections) or by the percutaneous, transmucosal,
transnasal or transpulmonary route, oral administration is also possible.

[0069]Peripheral neuropathy refers to a disorder of the nervous system not
including the brain and spinal cord. More specifically, it refers to a
peripheral nerve disease that involves disruption of myelin surrounding
nerve axons and eventual axonal degeneration. The disorder is observed
not only in nerve cells, but also in myelin-forming Schwann cells.
Peripheral neuropathy is associated with symptoms such as sensory
disturbance, numbness, hypoesthesia, ache, urtication, pain, dysesthesia,
anesthesia, dyskinesia, asthenia, paralysis (complete loss of movement),
etc.

[0070]The prophylactic and/or therapeutic agents of the present invention
are particularly useful for peripheral neuropathy which occurs when a
microtubule inhibitor is administered as an anticancer agent. A
microtubule, which is an intracellular structure, is composed of multiple
tubulins (polymerized tubulins). A tubulin dimer and a microtubule are in
a "dynamic equilibrium" state through polymerization and
depolymerization. An agent which disturbs this equilibrium state and
thereby exerts an anticancer effect serves as a microtubule inhibitor.
Those known as microtubule inhibitors include: 1) vinca alkaloids (which
have an inhibitory effect on microtubule formation through their binding
to tubulins to cause binding between tubulins), as exemplified by
vincristine, vinblastine, vindesine and vinorelbine; as well as 2) taxans
(which stimulate tubulin polymerization to stabilize microtubules, unlike
vinca alkaloids. As a result, taxans affect the formation and/or
functions of spindles (composed of microtubules) during cell division (M
stage) to stop the cell division, thereby causing cell damage, which
leads to an anticancer effect), as exemplified by paclitaxel (taxol),
taxotere and docetaxel.

[0071]The prophylactic and/or therapeutic agents of the present invention
can attain an excellent effect, particularly when used in combination
with an anticancer drug, such as paclitaxel or taxotere, which has a
strong anticancer effect and often causes neuropathy (e.g., numbness in
hands and feet) due to strong inhibition of microtubule action.

[0072]The present invention further provides the use of an
erythropoietin-containing pharmaceutical composition for the purpose of
cancer therapy, wherein the pharmaceutical composition is used in
combination with an anticancer agent having an inhibitory effect on
microtubules. Such anticancer agents having an inhibitory effect on
microtubules are preferably vinca alkaloids and taxans as mentioned
above, more preferably taxans. Particularly preferred are paclitaxel and
taxotere. The timing at which the erythropoietin-containing
pharmaceutical composition of the present invention is combined with the
above anticancer agent is determined by physicians in consideration of
the effect of erythropoietin as well as the disease to be treated and its
condition, the age and body weight of a patient, the route of
administration, etc. It may be any timing of before, simultaneously with,
or after administration of the above anticancer agent, particularly
preferably before or simultaneously with administration of the above
anticancer agent.

[0073]The present invention further provides a method for preventing or
treating peripheral neuropathy caused by microtubule inhibition, which
comprises administering a pharmaceutical composition containing a
prophylactically or therapeutically effective amount of erythropoietin to
a patient in need of such prevention or treatment.

[0074]The present invention further provides a method for cancer therapy,
which comprises administering to a patient a pharmaceutical composition
containing erythropoietin in an amount effective for preventing or
treating peripheral neuropathy caused by microtubule inhibition, wherein
the pharmaceutical composition is administered in combination with an
anticancer agent having an inhibitory effect on microtubules.

[0075]The present invention further provides the use of erythropoietin in
the manufacture of a pharmaceutical composition for preventing or
treating peripheral neuropathy caused by microtubule inhibition.

[0076]The present invention further provides a combined formulation, which
comprises a pharmaceutical composition containing erythropoietin in an
amount effective for preventing or treating peripheral neuropathy caused
by microtubule inhibition and an anticancer agent having an inhibitory
effect on microtubules.

[0077]In the present invention, an embodiment where an
erythropoietin-containing pharmaceutical composition is administered "in
combination with" an anticancer agent having an inhibitory effect on
microtubules covers all cases, i.e., before administration of the
anticancer agent, simultaneously with administration of the anticancer
agent, and after administration of the anticancer agent.

EXAMPLES

[0078]The following examples further illustrate the present invention
without, however, limiting the same thereto. Various changes and
modifications can be made by those skilled in the art, and these changes
and modifications are also included in the present invention.

Example 1

Effect of EPO in Taxotere-Induced Peripheral Neuropathy Model

<Experimental Materials and Methods>

(1) Preparation of Taxotere-Induced Peripheral Neuropathy Model

(1-1) Animals

[0079]ICR mice (male) were purchased at 5 weeks of age and handled for 1
week before use in the experiment.

[0081]Using the attached solution and physiological saline, taxotere was
adjusted to a volume of about 0.15 ml per 10 g mouse body weight (45
mg/kg iv, 90 mg/kg ip). The vehicle serving as a control (0 mg/kg) was
prepared in the same manner using Tween-80 and physiological saline for
iv administration.

[0082]On the other hand, recombinant human EPO under the trade name Epogin
(Chugai Pharmaceutical Co., Ltd., Japan) was adjusted using a solution
(containing 135 nmol/L NaCl, 0.125% histidine-HCl and 0.005% Tween 80 in
10 mmol/L phosphate buffer (pH 6.0)) and administered at 720 U/head/week.
The administration schedule was as follows: taxotere was given at 0 or 45
mg/kg iv (via the tail vein) or at 90 mg/kg ip, once a week for 3 weeks
(3 times in total on days 0, 7 and 14), while the vehicle and EPO (720
U/head) were each given sc (into the dorsal neck region) 3 hours before
taxotere treatment.

[0083]The mice were divided into the following 6 groups, with the number
of animals given in parentheses.

[0084]1. Taxotere 0 iv+Vehicle (5)

[0085]2. Taxotere 0 iv+EPO (5)

[0086]3. Taxotere 45 iv+Vehicle (8)

[0087]4. Taxotere 90 ip+Vehicle (8)

[0088]5. Taxotere 90 ip+EPO (8)

(2) Evaluation of Neurological Symptoms

[0089]Continuous treatment with taxotere will induce typical hind leg
crossing in mice when hanged by their tails. If the symptoms reach an
advanced stage, the same crossing response will also occur in the
forelegs, thereby leading to dysbasia. Such a symptom was used as an
indicator to evaluate the mice as follows: 0=no symptom; 0.5=abnormal
movement without clear flexion; 1=flexion in one hind leg; 2=hind leg
flexion or extension lasting for 2 seconds or longer; 3=dysbasia. Scoring
was accomplished by blind evaluation of symptoms.

(3) Histopathological Evaluation of Sciatic Nerves

[0090]Muscle tissue was peeled off to expose sciatic nerves. Under this
state, a glutaraldehyde fixing solution (stored on ice immediately before
use) was added to immerse all the sciatic nerves. After incubation for 10
minutes, the sciatic nerves were isolated and gently shaken for 1 hour in
a shaker containing a glutaraldehyde fixing solution. The sciatic nerves
were then embedded in a standard manner into a resin for electron
microscopic examination. Toluidine blue-stained sections were prepared
and evaluated for the degree of axonal degeneration at three levels: none
(-), mild (+) and moderate (++).

<Results>

(1) Neurological Symptom Changes

[0091]Upon taxotere treatment, neurological symptoms developed around 8
days after the treatment in both cases of 45 mg/kg iv and 90 mg/kg ip.
Then, the symptoms became further exacerbated day by day. Although the
symptoms tended to be exacerbated faster in the 90 mg/kg ip group than in
the 45 mg/kg iv group on days 7 to 10, these groups showed almost the
same course of symptoms and no significant difference over the subsequent
days (FIG. 1). In contrast, the group not receiving taxotere (naive)
showed no neurological symptom (FIG. 1). Based on these results, taxotere
treatment at 90 mg/kg ip was used for examination of drug effects because
it allowed faster development of the symptoms.

[0092]In neuropathy induced by taxotere treatment at 90 mg/kg ip, EPO
administration was found to reduce significantly the neurological
symptoms after 10 days (FIG. 2).

(2) Histological Examination

[0093]The results of histological examination by toluidine blue staining
are shown in Table 1 and FIGS. 3-1 and 3-2.

[0094]The groups not receiving taxotere (0 iv) showed no degeneration of
sciatic nerve axons, whereas the groups receiving taxotere showed
diameter reduction and deformation of axons, which were indicative of
clear axonal degeneration. The degree of axonal degeneration in the 90
mg/kg ip group was as follows: ±, n=3; mild (+), n=1; moderate (++),
n=3 (FIG. 3-2, Table 1). In contrast, the group receiving EPO showed the
degeneration levels within the range of ± to mild (+) levels even upon
90 mg/kg ip treatment, and the number of animals evaluated as the ±
level was larger than in the vehicle group, thus indicating a tendency to
alleviate neuropathy (FIG. 3-2, Table 1).

[0096]When a pharmaceutical composition comprising erythropoietin as an
active ingredient is administered to patients receiving a microtubule
inhibitor as an anticancer agent, such treatment alleviates peripheral
neuropathy in the patients and enables increased dosage, prolonged period
and increased frequency for administration of the above anticancer agent,
which were never before achieved. Thus, the pharmaceutical composition
not only contributes to improvement of QOL in the patients, but also
enables prolongation of life in the patients.